The expression of a DNA sequence in plants requires a promoter that is immediately upstream of the DNA sequence and is functional in plants. The properties of the promoter determine the sites and timing of expression in the plant. A promoter that is constitutively active will direct expression in all cells and tissues of the plants whereas a promoter that is active in a preferred manner in certain cells and tissues will correspondingly direct expression in those parts of the plant. The function of a promoter is dependent upon the presence of a core promoter that usually comprises a TATA box and a transcriptional start site that directs RNA polymerase to initiate synthesis of RNA at a particular position for a given DNA sequence. The promoter may also comprise additional sequences that are generally located 5′ but may also be located 3′ to the core promoter, and regulate expression from the promoter. These additional sequences may comprise nucleotide sequences that act as enhancers or suppressors. Enhancers and suppressors are the DNA sites through which transcription activator and repressor proteins respectively exercise their regulatory effects on transcription. Enhancers and suppressors are known in the art and include for example the 35S enhancer element. DNA sequences comprising the core promoter and regulatory sequences may be included in transformation vectors for expression of desired DNA sequences in plants including tissue-preferred expression.
A number of promoters including tissue-preferred promoters have been identified in plants [Venkataraman et al, (2004) Mol. Genet. Genomics 270(5): 378–86; Furtado et al, (2003) Plant Mol. Biol. 52(4): 787–99; Trindade et al, (2003) Gene 303: 77–87; Liu J J and Ekramoddullah (2003) Plant Mol. Biol. 52(1): 103–20]. These include a root-preferred promoter (U.S. Pat. No. 6,518,483 B1). In addition promoter elements and corresponding DNA-binding proteins that are restricted to particular plant tissues have also been identified [Yin et al, (1997) EMBO J. 16(17): 5247–59; Yanagisawa and Sheen (1998) Plant Cell 10(1): 75–89]. It is considered of agronomic importance to acquire the ability to drive tissue-preferred expression of genes of interest in transgenic plants. For instance increased resistance to soil borne or root pathogens might be achieved through transformation of a plant by a DNA sequence that directs expression of a pathogen resistance gene under the control of a root-preferred promoter. As another instance, improved tolerance of a plant to abiotic stress such as water or salt stress may be facilitated by expression of a gene conferring tolerance in a part of the plant such as the root that may be particularly important with respect to that stress. Alternatively as another instance, root-preferred expression of a gene that causes root proliferation and increased root density may lead to higher and more efficient nutrient uptake by the root system.
It may also be desirable to inhibit the expression of a native DNA sequence in a plant in order to produce a particular phenotype. This inhibition may be achieved by expression of an antisense or dsRNA that interferes with expression of the native DNA sequence. It may be desirable to direct this expression in a tissue-preferred manner using a tissue-preferred promoter. Thus the ability to direct tissue-preferred expression of a DNA sequence of interest requires the development of a collection of tissue-preferred promoters that would drive expression in different tissues. It is also recognized by those skilled in the art that a single tissue-preferred promoter may show variation in the strength and degree of specificity of tissue-preferred expression when introduced into the genomes of different plant species. Hence it is also considered desirable by those skilled in the art to develop access to multiple tissue-preferred promoters that differ in their DNA sequence but show similarity with respect to their pattern of tissue-preferred expression, as one such promoter may perform better in a particular plant species than another promoter. Therefore isolation and characterization of additional tissue-preferred promoters including root-preferred promoters is desirable in order to carry out genetic manipulation of plants of agronomic interest.
The identification of genes showing tissue-preferred expression in plants is a first step towards isolation of the corresponding promoter regions from the genes and characterization of the promoter to test for tissue-preference of expression at the level of transcription. Genes showing tissue-preferred expression have been identified in plants using several different experimental approaches including a) subtractive hybridization [Crossley et al, (1995) Planta 196: 523–529.] b) differential cDNA screening [Kim Hyun Uk and Chung Tae Young (1997) Plant Mol. Biol. 33 (1): 193–198] and c) differential display RT-PCR [Song and Allen (1997) Biochim. Biophys. Acta 1351: 305–312].
An alternative strategy to identify tissue-preferred pattern of gene expression involves the use of enhancer detectors [Bellen (1998) Plant Cell 11(12): 2271–2281] which comprise a mobile genetic element (transposon or T-DNA) carrying a reporter gene that contains a minimal promoter. Insertion of the transposon in the genome nearby an enhancer sequence may confer expression of the reporter gene in a tissue-preferred manner that reflects the activity of the nearby enhancer. Enhancer detection using engineered maize Ds transposon elements called enhancer traps has been successfully applied in Arabidopsis plants to detect genes that are expressed in a tissue preferred manner [Sundaresan et al, (1995) Genes and Development 9(14): 1797–1810]. The identification of an Enhancer trap line showing a desired pattern of tissue-preferred expression is a starting point for isolation and characterization of the corresponding promoter region responsible for the tissue-preferred expression. Herein is described the isolation and characterization of a promoter that confers root-preferred expression. The sequence was identified based on analysis of an enhancer trap line of Arabidopsis showing root-preferred expression. The Ds transposon insertion in the line is in the Arabidopsis gene At1 g73160 that encodes a putative glycosyl transferase.